SIBLING SPECIES, ADVERTISEMENT CALLS, AND REPRODUCTIVE ISOLATION IN FROGS
OF THE Leptodactylus pentadactylus SPECIES CLUSTER (AMPHIBIA, LEPTODACTYLIDAE)
W. R. Heyer,! R. O. de Sá,2 and A. Rettig2
Keywords: Leptodactylus pentadactylus species cluster, advertisement calls, reproductive isolation, speciation.
INTRODUCTION
A recent re-evaluation of morphological and adver- tisement call variation in the large species of frogs of the Leptodactylus pentadactylus cluster discovered more ex- amples of sibling species as defined by Ernst Mayr in his influential book Animal Species and Evolution. All previ- ously documented instances of sibling species in frogs demonstrated advertisement call differentiation consistent with the calls serving as pre-mating isolating mechanisms.
However, we find one instance of two species with non- distinguishable adult morphologies as well as non-distin- guishable advertisement calls. Presumably, the new in- stances of sibling species refiect retention of ancestral adult morphologies and advertisement calls. Larval and habitat differentiation appear to be important factors in the speciation processes in this group of frogs.
Ernst Mayr defined sibling species as, "morphologi- cally similar or identical natural populations that are re- productively isolated," in his influential book Animal Spe- cies and Evolution (Mayr, 1963 : 34). He considered the phenomenon to be important to understanding the com- plexity and scope of speciation processes in animals.
Mayr's concept of sibling species has been docu- mented in the frog genus Leptodactylus (Barrio, 1973;
Heyer et al., 1996). In these examples, reproductive isola- tion among sibling species was documented through anal- ysis of advertisement calls. That is, advertisement calls are very different from each other among species whose adults are morphologically identical. Females use the calls to dis- tinguish and select males of their own species to mate with in any mixed chorus where sibling species co-occur.
In the process of re-evaluating variation in the Lepto- dactylus pentadactylus species cluster, we discovered in- stances of sibling species that do not demonstrate repro- ductive isolation on the basis of advertisement calls.
' Amphibians and Reptiles, MRC 162, PO Box 37012, Smithsonian Institu- tion, Washington, DC 20013-7012, USA; E-mail: [email protected].
^ Department of Biology, University of Richmond, Richmond, VA 23173, USA; E-mail: rdesa(a!richmond.edu.
Herein we summarize the nature of the differentiation in- volved and discuss the evolutionary implications of our fmdings.
MATERIAL AND METHODS
Features for several color patterns, adult morphologi- cal characters, and measurements were analyzed in terms of geographic variation for the species currently known as Leptodactylus knudseni, labyrinthicus, and pentadactylus (Heyer et al., 2005). These data were used to postulate spe- cies boundaries for the taxa studied.
Molecular sequence data were obtained for 1807 base pairs of 12S and 16S mitochondrial rDNA genes using standard techniques (see Heyer et al., in press). The sequence data have GenBank accession numbers AY947855-82. Voucher specimen data are presented in the Appendix. The sequence data were analyzed with maxi- mum likelihood and parsimony methods using PAUP*
(Swofford, 1998).
RESULTS AND DISCUSSION Taxonomic Novelties
In order to discuss the biological implications of our results it is necessary to give a brief synopsis of some of our taxonomic findings. A set of specimens from the State of Para, Brasil identified in collections either as L. knudse- ni or L. labyrinthicus represent a new species. This new species will be referred to as the Para species for purposes of this paper. The taxon currently recognized as L. labyrin- thicus contains three species: L. labyrinthicus, L. vastus, and a new species from coastal Venezuela. Herein we use the available name L. vastus for the taxon occupying northeastern Brazil (justification in Heyer, in press). The taxon currently recognized as L. pentadactylus consists of four species: L. pentadactylus and three new species re-
3 >
> < >
1 1
o
1
2 1 0
-
A "> >A*^^^+++ +
o tí
-o tí
r/1
-1 - AA-^ rA<<\.^+%> -
-2 < ^ < A
-3 < -
<
-A
>
o
-13
-10
o
V ^^B K V e
V (j^^ fe o
+^ W Eä33<°
- A j ^4
^^^ ^ <ti < •
< •^< ^^^ \<^
^ ^ AI %
.^^
f^M^
/í +
• A A AZÍ
AY •
A
1 1 1 -10 5 0 5
First Canonical Variable O L hufonius X L elenae
+
L fuscasA L gracilis V L latinasus
< L mystacinus
10
^-3-2-10 1 First Canonical Variable C Middle America "pentadactylus"
• Pacific Ecuador "pentadactylus"
< L. pentadactylus -\- L. knudseni V Para
A L. labyrinthicus t> L. vastus
Fig. 1. Discriminant function analyses of male measurement data for comparable sets of species of the Leptodactylus pentadactylus species cluster (left) and the Leptodactylus fuscus group (right). The L.fuscus group species are those that occur together in the Gran Chaco of South America. Note that the data on the right are more structured than those on the left, for example, there is only a single cloud of points on the left and at least three clouds of points on the right and the sample confidence ellipses (p = 0.683) are much smaller on the right than the left. There are 6 confidence ellipses on the left (none for Pacific Ecuador "pentadactylus" with N=3) and 6 on the right (the ellipses for L. fuscus and L. latinasus are not visible due to small sizes of ellipses and density of data points).
ferred to herein as Middle American "pentadactylus" Pa- cific Colombia "pentadactylus," and Pacific Ecuador
"pentadactylus." The status of ¿. knudseni is unchanged.
We analyzed DNA fi-om all these species except for the coastal Venezuela and Pacific Colombia "pentadactylus"
species.
Lack of Adult Morphological
and Advertisement Call Differentiation
There is much less morphological differentiation among members of the L. pentadactylus cluster than found in other major clades of Leptodactylus studied to date. The measurement data illustrate the sort of differentiation ex- hibited by all the adult morphological data (Fig. 1). In par- ticular, adult morphologies can not distinguish L. knudseni from Middle American "pentadactylus" nor L. labyrinthi- cus, L. vastus, and the northern Venezuela species from each other (Heyer, in press).
The advertisement calls of L. knudseni can not be con- sistently differentiated from the calls of Middle American
"pentadactylus" (Fig. 2). The calls of these two species are
so similar to those of Z,. labyrinthicus that it is likely that females of any of these species would select males of the wrong species in a mixed chorus should they occur sympatrically.
Although there is no adult morphological or advertise- ment call differentiation among certain of the species, there are other features that are diagnostic. For example, L. knudseni and Middle American "pentadactylus" differ from each other in several larval internal buccal cavity characters as well as juvenile color patterns (Heyer, in press).
Genetic Relationsliips
The results of the mitochondrial rDNA gene analyses are best illustrated by the maximum likelihood bootstrap tree (Fig. 3) and the comparison of sequence differences among the samples analyzed (Table 1).
Contrary to expectations based on the morphological and advertisement call data, neither L. knudseni/Middle American "pentadactylus" nor L. labyrinthicus/vas tus are
kmihuitf^
100 msec
2-1
itHh! I
a 1
100 msec
0.2 Time, sec
0.4 0.1
Time, sec
0.2 0.3
Leptodactylus knudseni Middle American '•'•pentadactylus^'
Fig. 2. Wave forms and audiospectrograms oí Leptodactylus knudseni (USNM recording 228, cut 1) (left) and Middle American "pentadactylus"
(USNM recording 106, cut 1) (right). The calls have the same duration, structure, and overlapping broadcast frequencies.
sister-Species in tlie molecular cladogram (Fig. 3) that re- flects tlieir genetic relationsliips.
Sibling Species in Frogs Revisited
Our study demonstrates additional examples of sibling species as defined by Mayr (1963) in frogs of the genus Leptodactylus. Although sibling species in frogs are not common, they are not unexpected because they have been documented in several families of frogs. We did expect to find that advertisement calls of sibling species in frogs would serve as a reproductive isolating mechanism. Find- ing sibling species that do not differ in advertisement calls but demonstrate extensive molecular differentiation has not been previously reported in frogs that we are aware of.
There are two implications of our findings.
1) Ancestral adult morphologies and advertisement calls are retained in several species of the L. pentadactylus cluster. The fact that advertisement calls do not differ among some species of the L. pentadactylus cluster re- quires rethinking of the role of advertisement calls in the speciation processes of frogs.
2) Other factors than adult morphology and advertise- ment calls play an important role in speciation within the L. pentadactylus cluster There is much more larval varia-
tion within the L. pentadactylus cluster than observed in other major Leptodactylus clades. The variation includes differentiation in internal buccal cavity morphology, oral disk morphology, and aquatic versus terrestrial larvae (Heyer, in press). Habitat differentiation is pronounced among the species of the L. pentadactylus cluster in gen- eral and especially in those species lacking differences in adult morphology or advertisement calls (Heyer, in press).
For example, L. labyrinthicus is closely associated with the Cerrado Morphoclimatic Domain, L. vastus with the Caatinga Morphoclimatic Domain, whereas the morpho- logically similar Para species occurs only within the rain forests of the Amazonian Equatorial Morphoclimatic Do- main as defined by Ab'Sáber (1977).
Our study indicates that the role of advertisement calls in frog speciation processes requires re-evaluation. We have been able to identify this finding because we ana- lyzed three different data sets for the same taxa of frogs:
morphological, advertisement call, and genetic. We sus- pect that additional examples of sibling species lacking differentiation in advertisement calls will be found in the future with analyses of variation in morphology, advertise- ment calls, and molecules simultaneously on the same taxa.
85
96 • Para
98
100
100
10 changes
• L. vastus
L. labyrinthiciLS
• L. lahyrinthicus
100
53
100
91
Middle America
^'pentadactylus"
Middle America
^^pentadactylus"
• Middle America
^^pentadactylus"
100
100
• L. pentadactylus
L. pentadactylus
- Pasific Ecuador
^^pentadactylus'' Pasific Ecuador ''pentadactylus"
L. kitudsetti
Fig. 3. Maximum likelihood bootstrap analysis of combined 12S and 16S mitochondrial rDNA data. Numbers are bootstrap values greater than 50% support.
Caramaschi, Andrew Chek, Luis A. Coloma, Ronald I. Crombie, Jeremy Jacobs, and Miguel T. Rodrigues for allowing us to uti- lize tissue samples they collected.
George R. Zug (USNM) kindly reviewed the manuscript.
Financial support was provided by the Neotropical Low- lands Research Program, Smithsonian Institution (Richard R Vari, Principal Investigator) and the National Science Founda- tion (awards #9815787 and #0342918 and subsequent REU amendments) to RdS and WRH.
REFERENCES
Ab'Sáber A. N. (1977), "Os dominios morfoclimáticos na América do Sul. Primeira aproximaçào," Geomorfologia (Sao Paulo), No. 52, 1 - 22 + map.
Barrio A. (1973), ''Leptodactylus geminus una nueva especie del grupo fuscus (Anura, Leptodactylidae)," Physis, 32, 199-206.
Heyer W. R. (in press), "Variation and taxonomic clarification of the large species of the Leptodactylus pentadactylus spe- cies group (Amphibia: Leptodactylidae) from Middle Amer- ica, northern South America, and Amazonia," Arquivos de Zoología.
Heyer W. R., de Sa R. O., and MuUer S. (2005), "On the enig- matic distribution of the Honduran endemic Leptodactylus Silvanimbus (Amphibia: Anura: Leptodactylidae)," in: Don- nelly M. A., Crother B. I., Guyer •., Wake M. H., and White M. E. (eds.), Ecology and Evolution in the Tropics'. A Herpe- tological Perspective, Univ. of Chicago Press, Chicago, pp. 81-101.
TABLE 1. General Time-Reversible Distance Matrix
Lr Lk PI P2 ivl iv2 Ll\ Lll MApl MAp2 MAP3 PEl PE2 Lp\ Lpl
Lr
Lk 0.30
PI 0.30 0.14 -
P2 Lv\
Lvl Ll\
0.31 0.31 0.32 0.31
0.14 0.17 0.15 0.13
0.01 0.04 0.05 0.18
0.05 0.05 0.18
0.01 0.19 0.18
m
0.30 0.10 0.15 0.16 0.20 0.19 0.03 -MApl 0.34 0.09 0.15 0.15 0.19 0.18 0.12 0.10 -
MAp2 0.34 0.09 0.15 0.15 0.19 0.18 0.12 0.10 0.00 -
MAp3 0.36 0.12 0.18 0.18 0.19 0.18 0.08 0.14 0.03 0.03 -
PEl 0.34 0.12 0.18 0.18 0.21 0.19 0.16 0.12 0.07 0.07 0.12 -
PE2 0.34 0.12 0.18 0.18 0.21 0.19 0.16 0.13 0.07 0.07 0.11 0.00 -
Lp\ 0.35 0.11 0.21 0.22 0.25 0.23 0.16 0.13 0.06 0.06 0.10 0.09 0.09 -
Lpl 0.34 0.10 0.20 0.21 0.24 0.22 0.16 0.13 0.06 0.06 0.10 0.09 0.09 0.00 -
Lr, Leptodactylus rhodomystax, MZUSP 940333; Lk, Leptodactylus knudseni, QCAZ 13077; PI, Para species, MZUSP 70023; P2, Para species, MZUSP 70075; Lvl, Leptodactylus vastus, MTR 976629 (MZUSP ???); Lv2, Leptodactylus vastus, USNM 284552; Lll, Leptodactylus lahyrinthicus, USNM 303175; Lll, Leptodactylus lahyrinthicus, UC 233; MApl, Middle American '"pentadactylus" species, USNM 347153; MAp2, Middle Ameri- can "pentadactylus" species, USNM 298079; MAp3, Middle American "pentadactylu.<i" species, USNM 534219; PEl, Pacific Ecuador "pentadactylm"
species, QCAZ 17056; PE2, Pacific Ecuador "'pentadactylus" species, QCAZ 19859; Lpl, Leptodactylus pentadactylus, USNM 303466;
Lp2, Leptodactylus pentadactylus, MZUSP 70917.
Heyer W. R., Garcia-Lopez J. M., and Cardoso A. J. (1996),
"Advertisement call variation in the Leptodactylus mystaceus species complex (Amphibia: Leptodactylidae) with a de- scription of a new sibling species," Amphibia-Reptilia, 17, 7-31.
Levitón A. E., Gibbs Jr. R. H., Heal E., and Dawson C. E.
(1985), "Standards in herpetology and ichthyology: Part I.
Standard symbolic codes for institutional resource collec-
tions in herpetology and ichthyology," Copeia, 1985, 802 - 832.
Mayr E. (1963), Animal Species and Evolution, The Belknap Press of Harvard Univ. Press, Cambridge (MA), pp. xiv + 1 - 797.
Swofford D. L. (1998), PAUP*. Phylogenetic Analysis Using Parsimony (* and Other Methods). Version 4, Sinauer Assoc.
Sunderland (MS).
Appendix
Voucher specimen data used in molecular analyses. Museum designations follow Levitón et al. (1985) with the addition of Museo de Zoología de la Pontificia Universidad Católica del Ec- uador, Quito (QCAZ).
Leptodactylus knudseni. QCAZ 13077 • Ecuador, Fran- cisco Orellana, Parque Nacional Yasuni.
Leptodactylus labyrinthicus. Ulisses Caramaschi field num- ber 233 • Brasil, Sao Paulo, Piraju. USNM 303175 • Brasil, Sao Paulo, Fazenda Jataí, 5 km S of Luis Antonio.
Middle American '•^pentadactylus" species. USNM 298079, 347153 • Panama, Bocas del Toro, Isla Popa. USNM 534219 • Honduras, Colon, Quebrada Machin.
Pacific Ecuador ^pentadactylus'''' species. QCAZ 17056 • Ecuador, Esmeraldas, Alto Tambo. QCAZ 19859 • Ecuador, Esmeraldas, Bosque Protector La Perla.
Para species. MZUSP 70023 • Brasil, Para, Aldeia A-Ukre. MZUSP 70075 • Brasil, Para, Rio Vermelho.
Leptodactylus pentadactylus. MZUSP 70917 • Brasil, Para, Serra de Kukoinhokren. USNM 303466 • Brasil, Para, near Cachoeira do Espelho, ca. 50 km (airline) S of Altamira.
Leptodactylus rltodomystax (outgroup taxon). MZUSP 70375 • Brazil, Para, Serra de Kukoinhokren.
Leptodacytlus vastus. MTR 976629 (to be deposited in MZUSP) • Brasil, Mato Grosso, Gaucha do Norte.
USNM 284552 • Brasil, Pemambuco, near Caruaru.
